Plasmonic hot spots reveal local conformational transitions induced by DNA double-strand breaks

DNA double-strand breaks (DSBs) are typical DNA lesions that can lead to cell death, translocations, and cancer-driving mutations. The repair process of DSBs is crucial to the maintenance of genomic integrity in all forms of life. However, the limitations of sensitivity and special resolution of analytical techniques make it difficult to investigate the local effects of chemotherapeutic drugs on DNA molecular structure. In this work, we exposed DNA to the anticancer antibiotic bleomycin (BLM), a damaging factor known to induce DSBs. We applied a multimodal approach combining (i) atomic force microscopy (AFM) for direct visualization of DSBs, (ii) surface-enhanced Raman spectroscopy (SERS) to monitor local conformational transitions induced by DSBs, and (iii) multivariate statistical analysis to correlate the AFM and SERS results. On the basis of SERS results, we identified that bands at 1050 cm−1 and 730 cm−1 associated with backbone and nucleobase vibrations shifted and changed their intensities, indicating conformational modifications and strand ruptures. Based on averaged SERS spectra, the PLS regressions for the number of DSBs caused by corresponding molar concentrations of bleomycin were calculated. The strong correlation (R2 = 0.92 for LV = 2) between the predicted and observed number of DSBs indicates, that the model can not only predict the number of DSBs from the spectra but also detect the spectroscopic markers of DNA damage and the associated conformational changes.

. AFM imaging of pUC19 circular DNA plasmid damaged with bleomycin and fixed on mica: A) untreated DNA; B-I) DNA reacting for 4 mins with Fe(III)-bleomycin solution: linear fragments of various length are visible, the degree of fragmentation corresponds to increasing bleomycin concentration.     Since gold nanoparticles are more stable and inert (less prone to oxidation) in physiological buffer than silver 12 , gold nanoparticles were selected in these studies as the SERS substrate.
The optimization of SERS technique for efficient DNA measurements considered examination of quality (SNR) of spectra achieved with the use of three different types of gold nanoparticles: stabilized with sodium borohydride (Fig. S4 A), trisodium citrate (Fig. S4 B) and cysteamine ( Fig. S4 C). SERS spectra of nanoparticles with aggregating agent (0.05 M NaCl) and DNA were compared with the Raman spectra of concentrated DNA. For sodium borohydride nanoparticles, well-defined spectrum of stabilizer was obtained. Regarding that stabilizer itself is also clearly visible in the SERS DNA spectrum, these nanoparticles were not included in further measurements (Fig. S4 A). Comparing the Raman spectrum of concentrated DNA with SERS spectrum of DNA obtained with trisodium citrate nanoparticles, only one peak associated to phosphodiester bonds vibrations in DNA molecule at 734 cm -1 is well-resolved. As the SERS spectrum of DNA under selected conditions was not obtained, these nanoparticles were not considered in further studies as well (Fig. S4 B). For nanoparticles stabilized with cysteamine, well-resolved SERS spectrum of DNA was acquired with low influence of stabilizer itself (  Regarding the concentration of DNA in the sample, 114 mg L -1 was considered as the most appropriate due to the relatively low signal from stabilizer (the band from CH2 torsion at 1266 cm -1 ) in the acquired spectra ( Fig. S5 A). Whereas for the ionic strength measurements, the concentration of 36 mM NaCl was selected as the SERS spectra of DNA collected under these conditions are characterized by the best signal-to-noise ratio and the highest intensity of the characteristic DNA Raman marker bands (Fig. S5 B). Before establishing final experimental parameters, number of additional optimization measurements were performed. We also examined the effect of sequence of experimental steps, and various incubation times (Fig. S6).
Based on numerous systematic measurements, the most optimal experimental parameters were selected and then applied to the further studies of DNA damage.

Synthesis of nanoparticles
Cysteamine-stabilized gold nanoparticles (CHSBAuNPs) were prepared according to the modified Niidome et al. 16  The ultrafiltration process, conducted according to the protocol described by Oćwieja et al. 18 , was used for the purification of each type of obtained AuNP suspension.
Physicochemical characteristic of AuNPs dispersed in aqueous suspension.
The conductivity and pH of the stock AuNP suspension were measured using a multifunctional pH/conducto-meter (Elmetron). The concentration of AuNPs dispersed in the purified suspension was determined based on the density measurements described in detail elsewhere 19 .  Typical TEM micrograph of AuNPs was presented on Fig. S8 A. Analysing this image one can observe that cysteamine-stabilized AuNPs exhibit nearly spherical shape. Moreover, it was found that the AuNPs were fairly monodisperse (Fig. S8 B) and their average size was equal to 13±3 nm. This size value remains in agreement with the findings presented before 18 hence one can conclude that the preparation method of cysteamine-stabilized AuNPs is highly reproducible. and pH at the temperature of 25 o C showed that at pH 5.8 the aggregation process appeared for ionic strength higher than 10 -2 M (Fig. S9 A). Independently on the ionic strength, the aggregation process of AuNPs leading to formation of aggregates of an average size of 348±24 nm was observed under alkaline conditions (for pH higher than 8) (Fig. S9 A) Thereby, the aggregation process of AuNPs detected by DLS technique explained the reason of bathochromic shift of plasmon absorption maximum under alkaline conditions (Fig. S7 B).
The electrokinetic properties of AuNPs were evaluated using electrophoretic scattering technique (ELS). It was established that the AuNPs were positively charged in board range of ionic strength and pH. The zeta potential of AuNPs dispersed in the stock solution at pH 5.8 was equal to 54±2 mV (Fig. S9 B). This value was comparable with the data described previously 18 . Similarly, the drop of zeta potential values with an increase of ionic strength and pH (Fig. S9 B) was also observed. It is worth mentioning that the aggregates of AuNPs formed at ionic strength range between 10 -2 -5x10 -2 M were positively charged. with bleomycin ( Fig S10) and pUC19 plasmid exposed to UVC radiation as a damaging factor ( Fig S11). In both cases, damaging factors (BLM, UVC) induced DSBs (visible on AFM images), and conformational changes can be observed in the SERS spectra.
In the SERS spectra of pUC19 plasmid DNA exposured to UVC radiation, we have observed similar spectral changes as induced by BLM treatment including partial shift of the phosphate symmetric stretching from DNA backbone, which is a marker of DNA conformational change.
A comparison of the spectra acquired from control and irradiated DNA shows the intensity